The publications and other materials used herein to illuminate the background of the invention, and in particular to provide additional details relating to the invention, are referenced in the following text and are grouped in the appended list of references.
Transforming growth factor β's (TGFβ) are one of the few natural growth inhibitors which inhibit the growth of most epithelial cells. In contrast to other inhibitors, TGFβ is generally non cytotoxic, even at concentrations which are orders of magnitude above its effective concentration. TGFβ is the prototype for over 40 family members which share structural and functional similarities. The TGFβ superfamily plays an essential role in almost every aspect of cellular processes, including early embryonic development, cell growth, differentiation, cell motility, and apoptosis. See for example Yue and Mulder (2001); Sporn and Vilcek (2000); Massaque (1998); and Hartsough and Mulder (1997). The ability of TGFβ to potently inhibit the growth of many epithelial cells and solid tumors of epithelial origin, i.e. colon, breast, prostrate, lung, liver, ovarian, etc. is well established. However, many solid tumors develop resistance to the growth inhibitory effects of TGFβ. When cells become resistant to TGFβ, they no longer have the natural growth check provided by TGFβ and, as a result, may proliferate continually. Thus, TGFβ plays an important role in tumorigenesis and tumor aggressiveness. This resistance to TGFβ-mediated growth inhibition may occur at many levels. For example, the tumor cells may become unable to activate latent TGFβ (see for example, Wakefield et al. (1987); and Sporn et al., (1987), may display mutations in signaling receptors (Kim et al., 2000), or may develop defective TGFβ signaling pathways (Hartsough and Mulder, 1997).
TGFβ interacts with two major signaling receptors, designated RI and RII. The current working model for activation of the TGFβ receptors involves TGFβ binding by RII, which is constitutively phosphorylated. RII recruits RI into a complex (TβR complex) and phosphorylates RI in its GS domain. The complex then propagates the signal, presumably by interacting with and/or phosphorylating downstream cytoplasmic components (Wrana et al., 1994).
TGFβ RI and RII are mutated in human cancer cells (Massague, 1998). Mutations in TGFβ type II receptors are most common, particularly in human colon and gastric cancers; these are associated with microsatellite instability and a loss of TGFβ responsiveness (Markowitz et al., 1995). In addition, gross structural defects in the RII gene, as well as alterations in transcriptional repression of RII, contribute to a loss of TGFβ responsiveness (Kim et al., 2000). Although alterations in TGFβ RI receptors are less common, transcriptional repression of RI may be an alternative mechanism for subverting the growth inhibitory response to TGFβ. Other more subtle alterations in RI may also exist (Kim et al., 2000). However, if normal TGFβ receptors are expressed in cells with mutated receptors, TGFβ responsiveness does not always return. Thus, signal transduction pathways must also be altered.
Several types of signaling components have been described as participants in TGFβ signaling. These include protein kinase C (PKC), phospholipase C, protein phosphatase 1, Ras, several mitogen-activated protein kinases (Mapks), the Smads, and some of the Rho family members (Hartsough and Mulder, 1997; Mulder, 2000). Aside from the Ras/Mapk and Smad pathways, however, the generality and biological significance of the other components in the TGFβ pathway is unclear.
It has been demonstrated that TGFβ activates Ras within 3-6 min in untransformed epithelial cells that are TGFβ-sensitive, but not in cells that are TGFβ-resistant (Mulder and Morris, 1992). Several mitogen-activated protein kinases (Mapks) are also rapidly activated (within 5-30 min) after addition of TGFβ (Hartsough and Mulder, 1995; Frey and Mulder, 1997). The activation of some of the Mapks is sustained, indicative of nuclear translocation of the protein and subsequent regulation of transcription. The rapid kinetics for these effects indicate that they are direct, and not the consequence of the release of other growth factors that could subsequently regulate the Ras/Mapk pathways. It has also been shown that activation of Ras by TGFβ is required for the regulation of various cell cycle proteins in the nucleus (Yue et al., 1998). More recently, it has become clear that TGFβ activation of these pathways is required for transcriptional regulation of TGFβ itself, and the resulting autoproduction of TGFβ (Yue and Mulder, 2000).
The Smad superfamily are highly conserved proteins which include the Drosophila MAD and DAD proteins, three C. elegans proteins, a few xenopus proteins, and nine mammalian Smad isoforms (reviewed in Wrana and Attisano, 2000; Miyazono, 2000). Based upon accumulating evidence pertaining to the Smads, a general model for Smad function has been proposed. The so-called “receptor activated” RSmads (i.e., Smads 1-3 and 5) are phosphorylated after receptor activation and form a complex with the Smad 4 type components (common-partner Smad or co-Smad). Smad 4/dpc4 itself is not phosphorylated, but its heterocomplex formation with the other Smads is thought to mediate translocation of Smads to the nucleus and transcriptional activation.
Over the past years, progress has been made in identifying signal transduction components activated by TGFβ superfamily members. However, even though various signaling proteins have been analyzed, the cytoplasmic pathways regulated by TGFβ are still very poorly understood. It should be noted that discovery of TGFβ signaling components which are essential for mediating the biological responses of TGFβ has been difficult. For example, Smads were identified based upon homology screens using the Drosophila or C. elegans cDNAs. It is further noted that both the Smad and the Ras/Mapk pathways regulate transcriptional events. Clearly, TGFβ has functions in addition to transcriptional regulation. Thus, there is a need for identifying components which play a role in TGFβ signaling and/or regulate cellular events in addition to transcription.
Several potential TGFβ signaling components have been identified based upon their interaction with TGFβ receptors. Initial studies led to the identification of FKBP12 and the alpha subunit of farnesyl transferase as TGFβ RI interactors, TRIP-1 as an RII-interacting protein, and clusterin (also called apolipoprotein J) as a component that interacts with both TGFβ receptors. (See for example, Kawabata et al., 1995; Ventura et al., 1996; Reddy et al., 1996). Additional studies led to the discovery of TRAP-1 as an interactor of the constitutively active triple RI mutant (L193A, P194A, T204D) and STRAP (a WD-domain protein) as a component that associates with both RI and RII (See for example, Charng et al., 1998; Datta et al., 1998). Another WD domain protein that interacts with TGFβ RI was identified as the βα subunit of protein phosphatase 2A (See for example, Griswold-Prenner et al., 1998). However, the functional significance of these factors is, at present, unclear.
Various Smad-interacting proteins have also been identified, including SARA, which interacts with both Smads and TGFβ receptors (See for example, Tsukazaki et al., 1998). Several of the Smad-interacting factors have been shown to function as transcriptional repressors or co-activators (See for example, Massague and Chen, 2000; Hata et al., 2000). These factors address the transcriptional functions of TGFβ. However, they do not explain or provide insight into how TGFβ accomplishes the diverse activities which make it a multi-functional polypeptide. In addition, the alterations found thus far in these signaling components do not explain the diverse types of cancers that arise from a loss of growth control related to TGFβ or its signaling pathways. Thus, it is clear that there are additional TGFβ signaling components and pathways which are important to the complete understanding of how TGFβ asserts its growth suppressive and other functions inside cells and how these pathways are subverted to result in epithelial cancers.
Since TGFβ plays a critical role in such a vast array of human pathologies, any factor in one of TGFβ's signaling pathways is sure to have therapeutic potential for one or more of these diseases. These human pathologies include cancer, atherosclerosis, restenosis, arteriosclerosis, diabetic kidney disease, glomerulosclerosis, most progressive renal disorders, other sclerotic diseases, stroke, chronic inflammation, arthritis, hyperreactivity of the lymphoid system, asthma, periodontal disease, glaucoma, pulmonary fibrosis, other fibrotic diseases, scarring during wound healing, osteoporosis, neurodegenerative diseases, ischemic injury, encephalomyelitis, autoimmunity, immunodeficiencies, and other immune mediated-pathologies (See Sporn and Vilcek, 2000).
Of particular interest, TGFβ has been shown to be a tumor suppressor displaying true haploid insufficiency in its ability to protect against tumorigenesis (Tang et al. 1998). Many of the signaling components identified in the TGFβ signaling pathway have also been shown to function as tumor suppressors. Nothwithstanding new technologies, aggressive surgery, and modern chemotherapy, there has been little change in the survival of cancer patients having specific types of cancers over the past several years. The genetics of cancer is complicated, involving multiple dominant, positive regulators of the transformed state (oncogenes) as well as multiple recessive, negative regulators (tumor suppressor genes). Over 100 oncogenes have been characterize thus far. However, fewer than a dozen tumor suppressor genes have been identified, although the number is expected to increase significantly (Knudson, 1993).
The involvement of so many genes underscores the complexity of the growth control mechanisms that operate in cells to maintain the integrity of normal tissue. So far, no single gene has been shown to participate in the development of all, or even the majority, of human cancers. The most frequently mutated tumor suppressor gene is the p53 gene, mutated in roughly 50% of all tumors. The hope for a new generation of specifically targeted anti-tumor therapeutics may rest upon the ability to identify tumor suppressor genes that play important roles in the control of cell division. TGFβ receptor-interacting proteins or components of TGFβ superfamily member signaling pathways are likely to represent potential tumor suppressor genes.
Dynein is a molecular motor protein which mediates intracellular transport; it conveys its cargo along polarized microtubules (MTs) toward the minus ends. It is a massive multisubunit complex composed of heavy chains (HC's), intermediate chains (IC's), light intermediate chains (LIC's), light chains (LC's), and IC/LC-associated proteins (Hirokawa, 1998). The MT-binding domain is located within the HC's that form the globular heads and stems of the complex, whereas cargo-binding activity involves the IC's and several classes of LC's that associate at the base of the soluble dynein particle. Evidence also exists to suggest that the IC/LC complexes have a distinct and stable structure (King, 2000).
Dynein superfamily members appear to control various cell functions which include axonal transport, flagellar motility, organization of the mitotic spindle, distribution of late endosomes and lysosomes, the centrosomal localization of the Golgi complex, vesicular transport from early to late endosomes, the apical transport of Golgi-derived membranes in epithelial cells, the movement of phagosomes, and others (see for example, in Hirokawa, 1998). However, little is known about the regulation of the movement that the dynein motors drive. A large number of cytoplasmic dynein-associated proteins with diverse structural and functional roles have been identified (Milisav, 1998). However, it remains obscure as to the requirement by dynein of so many associated LC's, and LC/IC-associated proteins.
A new family of dynein-associated proteins which are both Drosophila roadblock (robl)-like, Chlamydomonas dynein light chain 7 (LC7)-like, and Drosophila bithoraxoid (bxd)-like have been described (Bowman et al., 1999). The original Drosophila bithoraxoid (bxd) gene of this family has been described in detail, but the transcripts are not encoded into protein (Lipshitz et al., 1987). Bxd is actually part of a huge bithorax gene complex (BX-C) which plays a role in segment development in Drosophila (Morata and Kerridge 1981; Smolik-Utlaut 1990; Stem, 1998). However, no functional studies have been reported with regard to bxd-like encoded proteins. km23 has 67% homology with robl and is identical to the class 1 mammalian robl-like proteins thought to play a role in cytoplasmic dynein functions. Accordingly, these results suggest that km23 is a dynein LC of the LC7 family that plays an important role in MT dynamics. Our data indicate that this is, indeed, the case. However, more importantly, our discovery of km23 as a TGFβ-receptor interactor and a dynein-interactor establishes the first connection between TGFβ-receptors and minus end MT dynamics, and indicates that such receptors can regulate dynein-mediated MT functions.
Although a recent report has described MTs as a cytoplasmic sequestering network for the Smads (Dong et al., 2000), these results again focus on the transcriptional functions of TGFβ. In this context, MTs appear to function by negatively regulating Smad transcriptional responses. However, no direct studies have been performed to determine how TGFβ may regulate MTs to induce the intracellular transport of specific proteins, and/or to mediate the diverse functions of TGFβ.
MTs are 25-nm tubule-like structures composed of alpha- and beta-tubulin heterodimers (reviewed in Hirokawa, 1998). Several parallel protofilaments composed of linearly arranged heterodimers form the MT wall, to which MT-associated proteins and motor proteins bind. MTs are polar structures with a fast-growing plus end and a minus end. They function as “rails” for the transport of organelles via MT-associated motor proteins such as dynein. MTs are important for many cellular processes, and play an important role in the generation and maintenance of epithelial cell polarity (Hofer et al., 1998). Outer arm dynein-associated proteins interact with dynein at the base of the dynein particle, as well as possibly with other proteins present in the cytoplasmic dynein complex (Bowman et al., 1999). However, it is unclear whether growth factors such as TGFβ can regulate dynein MT transport.
The sum total of distinct, yet interacting, signaling pathways that are activated simultaneously after TGFβ receptor activation, will play a significant role in both the normal growth of cells and in a variety of critical pathological conditions. Accordingly, identification of additional TGFβ signaling components and pathways will greatly advance our overall understanding of intracellular targets that dictate the fate of the organism. Thus, a need in the art exists for the discovery of TGFβ receptor-interacting proteins or TGFβ signaling components, and the polynucleotides encoding them, in order to provide new compositions which are useful in the diagnosis, prevention, and treatment of disorders associated with cancer and other diseases the development and progression in which TGFβ has been implicated.